Molecular Psychiatry (2008) 13, 858–872 & 2008 Nature Publishing Group All rights reserved 1359-4184/08 $30.00 www.nature.com/mp ORIGINAL ARTICLE Evidence for the involvement of the subunit GluR6 (GRIK2) in mediating behavioral displays related to behavioral symptoms of mania G Shaltiel1, S Maeng1, O Malkesman1, B Pearson1, RJ Schloesser1, T Tragon1, M Rogawski2, M Gasior2, D Luckenbaugh1, G Chen1 and HK Manji1 1Laboratory of Molecular Pathophysiology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA; 2Epilepsy Research Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

The 6 (GluR6 or GRIK2, one of the kainate receptors) resides in a genetic linkage region (6q21) associated with bipolar disorder (BPD), but its function in affective regulation is unknown. Compared with wild-type (WT) and GluR5 knockout (KO) mice, GluR6 KO mice were more active in multiple tests and super responsive to amphetamine. In a battery of specific tests, GluR6 KO mice also exhibited less anxious or more risk-taking type behavior and less despair-type manifestations, and they also had more aggressive displays. Chronic treatment with lithium, a classic antimanic mood stabilizer, reduced hyperactivity, aggressive displays and some risk-taking type behavior in GluR6 KO mice. Hippocampal and prefrontal cortical membrane levels of GluR5 and KA-2 receptors were decreased in GluR6 KO mice, and chronic lithium treatment did not affect these decreases. The membrane levels of other glutamatergic receptors were not significantly altered by GluR6 ablation or chronic lithium treatment. Together, these biochemical and behavioral results suggest a unique role for GluR6 in controlling abnormalities related to the behavioral symptoms of mania, such as hyperactivity or psychomotor agitation, aggressiveness, driven or increased goal-directed pursuits, risk taking and supersensitivity to psychostimulants. Whether GluR6 perturbation is involved in the mood elevation or thought disturbance of mania and the cyclicity of BPD are unknown. The molecular mechanism underlying the behavioral effects of lithium in GluR6 KO mice remains to be elucidated. Molecular Psychiatry (2008) 13, 858–872; doi:10.1038/mp.2008.20; published online 11 March 2008 Keywords: glutamate receptor 6 (GluR6); kainate receptors (KARs); mood-related behaviors; bipolar disorder (BPD); knockout mice (KO); lithium

Introduction behavior, increased goal-directed behavior, increased pursuit of pleasurable activities with potentially Bipolar disorder (BPD) (also known as manic-depres- painful consequences and substance abuse. Episodes sive illness) is one of the most severely debilitating of depression are characterized by depressed or medical illnesses, affecting the lives and functioning irritable mood accompanied by markedly diminished of millions worldwide.1 Emerging data suggest that interest or pleasure in everyday activities, psycho- BPD arises from the complex inheritance of multiple motor agitation or retardation, changes in body weight susceptibility .2–4 Phenotypically, it is a very or appetite, poor memory and concentration, fatigue complex disease in which patients alternate between and recurrent thoughts of death or suicide.1 episodes of mania and depression. Manic episodes There is a growing appreciation that BPD can be are characterized by euphoric or irritable mood, best conceptualized as a genetically influenced dis- thought disturbances, such as racing thoughts and order of synapses and circuits. It is thus noteworthy grandiosity, and behavioral symptoms, including that recent human genetic studies have identified hyperactivity, poor judgment, recklessness, aggressive GRIK2 (which encodes for GluR6, a kainite receptor implicated in synaptic plasticity) as a potential BPD 5 Correspondence: Dr HK Manji, Chief, Laboratory of Molecular susceptibility gene. Genetic linkage of BPD to Pathophysiology, National Institute of Mental Health, National 6q21 has been demonstrated in several Institutes of Health, Bldg. 35, Room 1C-912, 35 Convent Drive studies, and genome-wide significant linkage was MSC-3711, Bethesda, MD 20892, USA. recently established by meta-analysis.6–9 Buervenich E-mail: [email protected] Received 11 August 2007; revised 17 December 2007; accepted 20 and colleagues investigated the broad linkage peak on December 2007; published online 11 March 2008 6q and found that a specific haplotype, located on the GluR6 regulates mania-related behaviors G Shaltiel et al 859 regulatory region of the human GluR6 gene, was asso- behaviors in GluR6 KO mice, a series of biochemical ciated with BPD. In addition, very recent post-mortem studies were also conducted. studies demonstrate a reduction in GluR6 mRNA levels in the brains of BPD patients,10 thus pointing to GluR6 as a potential candidate gene in the patho- Materials and methods physiology of BPD. Animals A completely independent genetic study has also recently implicated the GRIK2 gene in a behavioral KAR KO mice were originated from 129/Sv and C57/ phenotype that may be associated with BPD.11 Bl6 background, and then backcrossed with 129Sv/Ev mice for at least 10 generations to provide an isogenic Intriguingly, the very same gene has been found to 25 confer sensitivity to treatment-emergent suicidal 129Sv/Ev strain. GluR6 KO, GluR5 KO and WT male mice, aged 8 weeks old, were single housed in an ideation in a large collaborative genetic study of ± 1 individuals with mood disorders (Sequenced Treat- animal room at a constant temperature (22 1 C) and ment Alternatives to Relieve Depression (STAR*D)).11 a 12-h light/dark cycle with free access to food and Individuals with variations in genes encoding GluR6 water. Female mice were not used in the studies and GluR3 (an AMPA receptor) were 15 times more to avoid the potential confounding effects of the likely to experience treatment-induced suicidal menstrual cycle on behavioral and neurochemical ideation. Although the neurobiologic basis of this measures. Mice were subjected to multiple tests in the following sequence designed from less to more very rare (but serious) side effect is unknown, it is 26 noteworthy that individuals with BPD—and poten- intrusive as recommended previously: open-field, tially those with a bipolar diathesis—are susceptible social interaction, elevated plus maze, forced swim, to antidepressant-induced dysphoric activation/agita- resident–intruder, saline–response and psychostimu- tion and potential suicidality.12,13 lant–response tests. The tests were performed at least Among the ionotropic glutamate receptors are the 24 h apart. Additional cohorts of mice were used for kainate receptor (KAR) family comprising five sub- other behavioral evaluations, such as passive avoid- units: GluR 5–7 and KA-1-2 (also named glutamate ance test, and for experiments with lithium treatment. receptor ionotropic kainate (GRIK) 1–5, respectively). Mice to be compared were tested concurrently in KARs are expressed in the areas of neuronal circuits the same room under dim illumination (30 lux) by involved in mood regulation.14,15 They have been the same testers between 10:00 and 18:00 hours. shown to bidirectionally regulate the release of gluta- All experimental procedures were approved by the mate at the mossy fiber to CA3 synapses,16 and Animal Use Committee of the National Institute of notably, they are also involved in long-term poten- Mental Health (NIMH) and were conducted according tiation induction at the hippocampus and the to the National Institutes of Health guidelines. amygdala.17 Moreover, it is now widely accepted that presynaptic KARs control the release of GABA Passive avoidance test (g-aminobutyric acid) in the hippocampus and The test was conducted as described previously by the amygdala.16,18 Furthermore, a growing body of El-Ghundi,27 with the modification of using GEMINI evidence demonstrates that KARs have metabotropic Avoidance System driven by in-house software. In actions in addition to their ionotropic activity that brief, all mice were given one habituation trial to modulates both hippocampal inhibitory19 and excita- explore both chambers for 120 s, followed by two tory postsynaptic currents20 as well as pyramidal training trials separated by 5 min. During the training cell excitability.21,22 The metabotropic actions include trials, individual mice were placed in the bright interaction with G , induction of second chamber for 30 s, after which a guillotine door was messengers and activation of PKA ( kinase A), opened and the latency to enter the dark chamber was PKC (protein kinase C) and MAP (mitogen-activated measured for 300 s. On the second trial, on entrance protein) kinases.19–22 into the dark compartment (all four paws and In view of the GluR6 receptor as a putative BPD tail inside), the guillotine door was closed and the susceptibility gene, we undertook a series of animal mouse was confined in the dark compartment for studies to elucidate the role of this receptor in modu- 10 s, followed by two consecutive 3-s (inescapable) lating behavioral patterns related to facets of the foot shocks (0.6 mA), left in the compartment for an complex symptoms of BPD. Furthermore, to deter- additional 10 s, and then returned to their home cage mine potential specificity of this receptor to mood- and left there undisturbed until the next acquisition related behaviors, we also investigated the role of trial. Acquisition (learning) and retention (memory) GluR5 receptors. We used a battery of behavioral of passive avoidance response were assessed 5 min, studies tailored to monitor diverse symptoms of 24 h, 48 h and 14 days, respectively, after the training. either mania23 or depression.24 The chronic effects During assessment, each individual mouse was of lithium—the classic antimanic mood stabilizer— placed in the light chamber, and the latency to enter on behavioral alterations of mutant mice were also the dark chamber was recorded for up to 300 s. investigated. Finally, to assess any potential bio- Passive avoidance response was assessed by compar- logical changes associated with GluR6 ablation or a ing the step-through latencies during training and possible biochemical mechanism mediating aberrant assessment trials.

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 860 Open-field test 30 Â 5 cm with a 5 Â 5 cm center area, and the walls The experiment was carried out as described pre- of the closed arms were 40 cm high. Individual mice viously.28 In brief, a 50 Â 50 cm arena was used for the were placed in the center of the maze, tracked for open-field test. To study spontaneous locomotion, 5 min with a video camera, and then returned to their mice were placed in one corner of the arena and their home cage. The plus maze was wiped clean between behavior was recorded for 60 min with the Ethovision trials with a 10% alcohol solution. Time spent in the video tracking system (Noldus, Leesburg, VA, USA) maze and frequency of visits to the different zones and videotaped. The experiment was repeated for 3 of the maze were scored using Ethovision (Noldus) or consecutive days and again 14 and 15 days after the the TopScan Suite of CleverSystems (CleverSys Inc.). first exposure to the arena to test persistency of the activity pattern. To study saline injection response as Forced swim test a control for amphetamine injection, mice were first The test was conducted as described previously.28,30 placed in one corner of the arena and their behavior Briefly, transparent Plexiglas cylinders, 50 cm high was recorded for 30 min; they then received saline with a diameter of 20 cm, were filled with tap water at injection (2 ml kgÀ1, i.p.), were placed back to the 22–24 1C to approximately 25 cm in a way that mice same arena and their behavior was recorded for an were not able to touch the floor or escape. Individual additional 30 min. To test amphetamine response, the mice were placed in the water for a 6-min session, and same procedure was carried out, substituting amphe- their behavior was videotaped for later analysis. tamine (2 mg kgÀ1, Sigma, St Louis, MO) for saline. At the end of each session, mice were dried with a Measures of locomotor activity, total time spent in the paper towel and returned to their home cage. Water center and frequency measures for center visits (30 Â was replaced after each trial. The time of immobility, 30 cm) were collected. The open field was wiped defined as a lack of activity except movements between trials with a 10% alcohol solution. needed to keep their nose above water, was scored during the last 4 min of the session. Home cage activity Mice were individually housed in the new home cage Resident intruder test at the beginning of the testing period. After 1 day, A 129Sv/Ev naive male mouse was introduced into mice were transported in the same cage and placed in the home cage of each mouse tested. The interaction the testing room from 08:00 and 10:00 hours every day between the two mice was videotaped for 5 min, and for 2 consecutive days to avoid the potential effects of the frequency of attacks on the intruder was counted transportation and testing room on behavior. Activity in each trial. Each naive mouse was used no more of the mouse was then videotaped with a digital than once as an intruder. camera for 30 min between 08:00 and 10:00 hours every day for the following 2 consecutive days. Digital Lithium treatment video files were analyzed using CleverSystems’ The treatment was conducted using a previously HomeCageScan Suite (CleverSys. Inc., Reston, VA, tested regimen that maintains the trunk blood level USA). To validate the software, behavioral events of lithium within the human therapeutic range identified by HomeCageScan were compared with the (0.4–1.2 mM).1 WT and GluR6 KO mice were fed with findings of observers; between the software and the lithium carbonate chow (2.4 g kgÀ1, Bio-Serv, French- observers, consistency of each reported behavior town, NJ, USA) or control chow identical to lithium exceeded 90%. For the purpose of this study, two carbonate chow with the exception of lithium salt for types of home cage activities were scored: general 4–6 days (short-term treatment) and 4 weeks (chronic behavior (turning, walking, digging, grooming, drink- treatment). Brain lithium levels after chronic treat- ing and eating) and exploratory behavior (rearing, ment were 0.55±0.01 mM (mean±s.e.m.). Owing to sniffing and foraging). the well-known lithium side effects of polyuria and polydipsia, cage bedding was changed twice a week Social interaction test and animals were supplied with 0.9% NaCl (saline) Pairs of mice of the same genotype were placed in solution in addition to tap water to prevent possible opposite corners of a 50 Â 50 cm open-field arena. electrolyte imbalance. Their activities were videotaped for 5 min, and the frequency of social behaviors was scored for olfactory Immunoblotting investigations (sniffing), side-by-side behavior, tail Cytosolic and membrane fractions were prepared rattling and attack biting. Each arena was wiped clean using a previously described method.31 In brief, the between trials with a 10% alcohol solution. frontal cortex (FCX) and hippocampus samples were homogenized in a hypotonic protein extraction buffer Elevated plus-maze test containing a protease inhibitor cocktail (Sigma) and a The procedure was carried out as reported pre- phosphatase inhibitor cocktail I and II (Sigma) by viously.28,29 A Plexiglas plus-shaped maze containing passing through a 19-gauge needle 10 times, followed two dark and enclosed arms and two open and by a 22-gauge needle 10 times. The homogenates lit arms, elevated 50 cm above ground, was used to were then centrifuged at 1200 r.p.m. for 12 min at examine anxiety-related behaviors. The arms were 4 1C to remove non-dissolved debris and nuclear

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 861 components. The particulate (representing largely cell 300 WT membrane) and soluble (representing largely cyto- GluR6 KO plasmic) fractions were then separated by centrifuga- tion at 14 000 r.p.m. for 30 min at 4 1C. To make it 200 soluble, the membrane pellet was suspended in the protein extraction buffer added with 1% Triton X-100. 100 Protein concentrations were determined using BCA Protein Assay Kit (Pierce Biotechnology, Rockford,

IL). The linearity of the protein concentration for Step-through latency (s) 0 immunoblotting was ascertained by resolution of 5 min Day 1 Day 2 Day 15 selected concentrations of protein. Protein immunoblotting was conducted using pre- Pre-shock Post-shock 32 viously described methods. In brief, 5–10 mg protein Figure 1 GluR6 KO mice have no significant impairment samples were separated by 10% sodium dodecyl in the passive avoidance test. Acquisition (learning) and sulfate-gradient gel electrophoresis (Invitrogen, retention (memory) of the passive avoidance response did Carlsbad, CA) and immunoblotted with anti-GluR5 not differ in GluR6 KO and WT mice. During the training (1:500; Millipore, Billerica, MA), anti-KA-1 (1:1000; trial, all mice promptly entered the dark compartment, Abcam, Cambridge, MA), anti-KA-2 (1:1000; Upstate), whereas after receiving foot shocks, all mice quickly learned anti-GluR1 (1:1000; Millipore), anti-GluR2 (1:1000; to avoid entering the dark chamber (previously paired with Zymed Laboratories, San Francisco, CA), anti-GluR3 foot shock), as indicated by increased latencies during the post-shock test trials. No statistical difference was found (1:1000; Chemicon), anti-NR1 (1:500; Chemicon) or between the two groups in the avoidance latencies in the anti-NR2A (1:500; Upstate) antibodies. Anti-rabbit pre-shock trial and during retention trials conducted or anti-mouse IgG, horseradish peroxidase-linked 5 min, 1 day, 2 days and 15 days post-shock (two-way antibodies (Cell Signaling Technology, Danvers, MA) ANOVA: genotype effect, F1,50 = 2.75, P > 0.1 (NS)). Results were used as secondary antibodies. The immuno- are means±s.e.m. reactive bands were visualized by enhanced chemi- luminescence (Amersham Biosciences, Piscataway, to the novel environment, as indicated by gradual NJ) and exposed to Kodak BioMax or Biolight film movement reduction in the arena (Figure 2a). Com- (Eastman Kodak, Rochester, NY). The signal intensi- pared with GluR5 KO and WT mice, GluR6 KO mice ties were quantified using Kodak Imaging System, displayed increased spontaneous locomotor activity based on standard curves of each protein. (two-way ANOVA, genotype effect, F2,35 = 87.55, P < 0.0001) in a pattern consistent with enhanced Statistical analysis exploratory behavior rather than locomotor unrest. Two- and one-way analysis of variance (ANOVA), That is, if the GluR6 KO mice were simply hyperlo- post hoc Fisher LSD (least significant difference) and comoting, they would be expected to maintain similar Student’s t-tests were conducted using the software levels of activity throughout the trial. When introdu- STATISTICA 7 (Statsoft, Tulsa, OK, USA) and Prism 4 cing the animals to the same arena in repeated tests, (GraphPad Software Inc., San Diego, CA, USA). GluR5 KO and WT mice showed reduced activity; in contrast, the GluR6 KO mice continued to display hyperactivity in the same arena for at least 15 days Results (Figure 2b) (two-way ANOVA: genotype F2,174 = 148.3, Previous studies have shown that GluR6 KO mice P < 0.0001; day F4,174 = 4.9, P < 0.001; genotype  day have no gross neuroanatomical abnormalities or F8,174 = 1.2, P = NS (nonsignificant) and one-way AN- 33,34 sensorimotor deficits. GluR6 KO mice frequently OVA (day): for GluR6 KO mice, F4,60 = 1.0, P = NS; for engage in fighting within their home cages; therefore, GluR5 KO mice, F4,60 = 4.6, P < 0.01; for WT mice, mice of all genotypes were individually housed F4,54 = 3.6, P = 0.01). throughout these experiments. As previously shown Home cage activity monitoring showed that GluR6 by others,33 GluR6 KO mice weighed less (24.2± KO mice displayed increased home cage general 0.52 g (mean±s.e.m., n = 13)) than GluR5 KO (25.86± activity (Figure 2c) (two-way ANOVA: genotype 0.52 g (n = 13)) and WT mice (27.47±0.54 g (n = 12)) F1,14 = 8.194, P = 0.0125; time F1,14 = 1.926, P = 0.1869;

(ANOVA, F2,3 = 9.57, P < 0.001). Consistent with a lack genotype  time F1,14 = 0.02712, P = 0.8715) as well as of spatial learning and memory impairment,33,34 no increased exploratory activity (Figure 2d) (two-way significant effects of genotype were detected on the ANOVA: genotype F1,14 =10.99, P = 0.0051; time outcome measures of the passive avoidance test F1,14 =1.279, P = 0.2771; genotype  time F1,14 = 0.3059, (Figure 1), a test that assesses fear-associated learning P = 0.5890). and memory. GluR6 KO mice were more responsive to amphetamine GluR6 KO mice exhibited more spontaneous activity Saline injection alone did not change the activity The spontaneous activities of mice were examined in levels in any of the three groups of mice (Figure 3a). the open-field test and in their home cage. In the For mice of all three genotypes, amphetamine injec- open-field test, all three groups displayed habituation tion increased locomotor activity above the activity

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 862 1800 * * WT * * WT 7500 * 1500 GluR6 KO GluR6 KO GluR5 KO 1200 GluR5 KO 5000 900

600 2500 300 Distance traveled (cm) Distance traveled 0 0 0 10203040506070 (cm) distance traveled Total 1231415 Time blocks (min) Days of trial

WT 600 * 175 * WT GluR6 KO GluR6 KO 500 * 150 * 400 125 100 300 75 200 Frequency of Frequency

Frequency of Frequency 50 general behaviour general

100 behaviour general 25 0 0 Day 1 Day 2 Day 1 Day 2

Figure 2 GluR6 KO mice exhibit increased spontaneous activity in the open field and home cage. (a) To study spontaneous locomotion in the open field, mice were placed in one corner of an open-field arena and their behavior was recorded for 60 min. GluR6 KO mice displayed increased spontaneous locomotor activity during 1 h of the first day of the open-field test

compared with GluR5 KO and WT mice (two-way ANOVA, genotype effect, F2,35 = 87.55, P < 0.0001). (b) GluR6 KO mice displayed persistent hyperactivity in the open-field arena at each trial conducted over 3 consecutive days and also at days 14

and 15 from the first exposure (two-way ANOVA: genotype F2,174 = 148.3, P < 0.0001; day F4,174 = 4.9, P < 0.001; genotype  day

F8,174 = 1.2, P = NS and one-way ANOVA (day): GluR6 KO F4,60 = 1.0, P = NS; GluR5 KO F4,60 = 4.6, P < 0.01; WT F4,54 = 3.6, P = 0.01). To study the home cage activity, WT and GluR6 KO mice were monitored in their home cages using a digital camcorder for 1 h between 0800 and 1000 hours for 2 consecutive days. Digital video files were analyzed using CleverSystems’ HomeCageScan Suite for general activity (including, turning, walking, digging, grooming, drinking and eating) and exploratory activity. (c) GluR6 KO mice displayed significantly more home cage general activity, which was

consistent over 2 days of testing (two-way ANOVA: genotype F1,14 = 8.194, P = 0.0125; time F1,14 = 1.926, P = 0.1869;

genotype  time F1,14 = 0.02712, P = 0.8715). (d) GluR6 KO mice also displayed significantly more home cage exploratory

activity, which was consistent over 2 days of testing (two-way ANOVA: genotype F1,14 = 10.99, P = 0.0051; time F1,14 = 1.279, P = 0.2771; genotype  time F1,14 = 0.3059, P = 0.5890). Results are means±s.e.m. *Pp0.05.

level before the injection (Figure 3b). The ampheta- intruder test (Figure 4b), GluR6 KO mice attacked the mine-induced increases in locomotion were signifi- intruder significantly more frequently than WT mice

cantly higher in GluR6 KO mice (Figure 3c) (ANOVA (Student’s t-test: t23 = À2.12, P < 0.05). for genotype, F2,32 = 5.529, P = 0.0087; Tukey’s multi- ple comparison test, for WT vs GluR6 KO mice, GluR6 KO mice exhibited less anxious and more P < 0.05; for GluR5 KO vs GluR6 KO mice, P < 0.05). risk-taking behavior To measure anxiety- or risk-taking-related behavior in GluR6 KO mice were robustly aggressive the mice, center activity in the open-field test (Figure As mentioned before, there were more home cage 5) and open- or closed-arm activity in the elevated fights among GluR6 KO mice. To further assess social plus-maze test (Figure 6) were monitored. In these behavior and aggressiveness in these mice, the social tests, the amount of time spent in the anxiety- interaction and resident–intruder tests were con- provoking spaces (center of the open field and open ducted. In the social interaction test (Figure 4a), the arms of the maze) is thought to be driven by curiosity number of side-by-side events did not differ between and prohibited by fear and self-protection, and are the groups. Compared with GluR5 KO and WT mice, sensitive to treatment with anxiolytic drugs.36,37 GluR6 KO mice exhibited a significantly higher GluR6 KO mice exhibited significantly increased

frequency of sniffing, which is an index of investiga- entries to (Figure 5a) (one-way ANOVA: F2,35 = 33.6, tional activity (ANOVA, F2,19 = 3.44, P = 0.05; post hoc P < 0.0001) and time spent in (Figure 5b) (one-way Fisher LSD test: GluR6 KO vs WT mice, P < 0.03). ANOVA: F2,35 = 13.4, P < 0.0001) the center of the open GluR6 KO mice displayed significantly more attack field compared with GluR5 KO and WT mice. In the

bites (ANOVA, F2,19 = 3.9, P < 0.04) and events of tail elevated plus-maze test, GluR6 KO mice exhibited rattling (ANOVA, F2,19 = 11.4, P < 0.001); these are proportional (open arm vs all arms) increases in open- thought to reflect a state of high arousal and arm duration (Figure 6a) (ANOVA: F2,16 = 5.783, characteristic of dominant mice.35 In the resident– P = 0.0129; WT vs GluR6 KO mice, P < 0.05; WT vs

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 863 1800 i.p. saline 2700 WT 2400 1500 i.p. amphetamine GluR6 KO 2100 1200 1800 GluR5 KO WT 1500 900 GluR6 KO 1200 GluR5 KO 600 900 600 300 Distance traveled (cm)

Distance traveled (cm) 300 0 0 0102030 40 50 60 70 01020 30 40 50 60 70 Time blocks (min) Time blocks (min)

** 6000 5000 4000 3000 2000 1000

after i.p. amphetamine 0 above 25–30 min bin levels Increases in distance traveled WT

GluR6 KO GluR5 KO Figure 3 GluR6 KO mice exhibit increased amphetamine-induced response. (a) To study injection-induced irritability, mice were first placed in one corner of the arena and their behavior was recorded for 30 min as a control for amphetamine injection; immediately afterwards they were intraperitoneally injected with saline and placed back in the same arena for an additional 30 min. Saline injection did not affect locomotor activity for any of the three groups. (b) To study amphetamine- induced hyperactivity, the same procedure as in panel (a) was carried out, except that instead of saline, 2 mg kgÀ1 amphetamine was administered. Amphetamine increased locomotor activity above 25–30 min activity levels in mice of all three genotypes. (c) GluR6 KO mice displayed significantly more increases above the 25–30-min activity level after amphetamine injection compared with WT and GluR5 KO mice (ANOVA for genotype, F2,32 = 5.529, P = 0.0087; Tukey’s multiple comparison test, WT vs GluR6 KO mice, P < 0.05; GluR5 KO vs GluR6 KO mice, P < 0.05). Results are means±s.e.m. *Pp0.05.

18 WT 16 GluR6 KO 6.0 * 14 GluR5 KO 12 * * 4.5 10 * * 8 3.0 6 4 1.5 Attack frequency Distance traveled (cm) 2 0 0.0 Tail rattle Attack bite Sniffing Side by side WT GluR6 KO

Figure 4 GluR6 KO mice display robustly aggressive behavior. (a) In the social interaction test, pairs of mice within the same strain were placed in opposite corners of an open-field arena. The frequency of social behaviors was counted over 5 min. GluR6 KO mice displayed a high frequency of destructive (aggressive) behaviors compared with GluR5 KO and WT mice (one-way ANOVA for ‘tail rattling’: F2,19 = 11.4, P < 0.001; ‘attack bite’: F2,19 = 3.9, P < 0.04). GluR6 KO mice displayed a high frequency of exploratory behavior compared with WT mice (one-way ANOVA for ‘sniffing’: F2,19 = 3.44, P = 0.05; post hoc Fisher LSD test for ‘sniffing’: GluR6 KO vs WT mice, P < 0.03). GluR6 KO mice displayed the same side-by-side behavior as GluR5 KO and WT mice (one-way ANOVA for ‘side by side’ F2,19 = 0.02, P = NS). (b) In the resident–intruder test, a 129Sv/Ev naive male mouse was introduced to the home cage of each mouse tested. The frequency of attacks on the intruder was counted over 5 min. GluR6 KO mice exhibited a high frequency of attack events toward their intruder compared with WT mice, who exhibited no attacks toward their intruder (Student’s t-test: t23 = À2.12, P < 0.05). Results are means±s.e.m. *Pp0.05.

GluR6 KO mice, P < 0.05), entry frequency (Figure 6b) GluR6 KO mice displayed less immobility in the forced

(ANOVA: F2,16 = 6.256, P = 0.0098; WT vs GluR6 KO swim test mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05) and The forced swim test, a test for the behavioral effects distance traveled (Figure 6c) (ANOVA: F2,16 = 7.264, of antidepressants, is thought to monitor behavioral P = 0.0057; WT vs GluR6 KO mice, P < 0.05; WT vs despair in a goal-directed task (for example, escap- GluR6 KO mice, P < 0.05). ing).38 In this test, GluR6 KO mice displayed reduced

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 864 immobility time compared to the mice of other strains reflects hyperactivity, increased goal-directed beha-

(Figure 7) (one-way ANOVA: F2,35 = 4.5762, P < 0.05). vior or both, cannot be fully distinguished by this test Whether this reduced immobility in GluR6 KO mice alone.

175 Behavioral effects of chronic lithium treatment on 150 GluR6 KO and WT mice 125 Chronic lithium treatment relieves symptoms in manic patients, but does not alter mood state in 100 healthy subjects.1 Lithium, alone or as an adjunct to 75 antidepressant therapy, is also used to treat depres- 1 50 sion. To determine if the behavioral excitement and aggression observed in the GluR6 mice were respon- 25 sive to lithium, WT and GluR6 KO mice were treated

Frequency of center entries 28 0 with lithium using a clinically relevant regimen. WT GluR6 KO GluR5 KO The effects of short-term lithium treatment (4–6 days) were investigated. In WT mice, no significant beha- 1500 vioral effects of chronic lithium treatment were * * 1250

1000 150 * * 750 125

500 100 Center duration (s) 250 75

0 50

WT GluR6 KO GluR5 KO Immobility time (s) 25 Figure 5 GluR6 KO mice show increased activity in anxiety-provoking subregions of the open-field test. To 0 WT GluR6 KO GluR5 KO further study the activity in an anxiety-provoking environ- ment, mice were placed in one corner of an open-field Figure 7 GluR6 KO mice displayed decreased immobility arena; the frequency of entering events to the center of time in the forced swim test. Mice were placed in cylinders the arena and the total time spent in it was recorded filled with water in a way that they were not able to touch over 60 min. (a) GluR6 KO mice displayed a higher the floor or escape for 6 min. Immobility time, defined as a frequency of entries to the center of the open-field arena lack of activity aside from small movements needed to keep compared with GluR5 KO or WT mice (one-way ANOVA: the body floating, was measured throughout the last 4 min F2,35 = 33.6, P < 0.0001). (b) GluR6 KO mice increased their of the session. Immobility time was reduced for GluR6 KO time spent in the center of the open-field arena compared mice compared with GluR5 KO or WT mice (one-way

with GluR5 KO or WT mice (one-way ANOVA: F2,35 = 13.4, ANOVA: F2,35 = 4.5762, P < 0.05). Results are means±s.e.m. P < 0.0001). Results are means±s.e.m. *P < 0.0001. *P < 0.05.

75 75 75

50 50 50

25

Durations 25 25 Distances Frequencies

(ratios of open vs total) 0

0 (ratios of open vs total) 0 (ratios of open vs total) T T T W W W 6 KO 5 KO 6 KO 5 KO 6 KO 5 KO luR luR luR luR luR luR G G G G G G Figure 6 GluR6 KO mice exhibited increased activity in anxiety-provoking regions of the elevated plus maze test. A plus- shaped maze elevated above ground containing two dark closed arms and two open and lit arms without walls was used to examine activity in anxiety-provoking regions. Each mouse was placed in the center of the maze; time and frequency of visits to the different zones of the maze, as well as locomotion measures, were collected for 5-min sessions. (a) Ratios of open-arm vs total (open and closed arms) duration of time spent. GluR6 KO mice exhibited increases in duration of time spent in the

open arms of the maze (ANOVA: F2,16 = 5.783, P = 0.0129; WT vs GluR6 KO mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05).

(b) Ratios of open-arm vs total arm entries. GluR6 KO mice exhibited increased open-arm entries (ANOVA: F2,16 = 6.256, P = 0.0098; WT vs GluR6 KO mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05). (c) Ratios of open-arm vs total arm distance

traveled. GluR6 KO mice exhibited increased distance traveled in the open arms (ANOVA: F2,16 = 7.264, P = 0.0057; WT vs GluR6 KO mice, P < 0.05; WT vs GluR6 KO mice, P < 0.05). Results are means±s.e.m. *P < 0.05.

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 865 550 WT 500 WT+Li 200 450 GluR6 KO ** 175 GluR6 KO+Li 400 ** 150 350 ** 125 300 250 100 200 75 Distance traveled 150 50

100 Distance traveled (% of 25 50

(% of WT mice treated with control chow) 0 0 I II III GluR6 KO mice treated with control chow) Control Lithium Trials Figure 8 Chronic lithium treatment reduced spontaneous locomotor activity in GluR6 KO mice. (a) Four weeks of lithium treatment attenuated the spontaneous locomotor activity of GluR6 KO mice studied over 3 consecutive days of open-field test

(two-way ANOVA: genotype F3,57 = 647.7, P < 0.0001; day F2,57 = 1512, P = NS; genotype  day F6,57 = 0.8, P = NS; one-way

ANOVA: day 1: F3,19 = 7.7, P < 0.01; day 2: F3,19 = 25.7, P < 0.001; day 3: F3,19 = 14.2, P < 0.001; post hoc Fisher LSD test: P < 0.05, P < 0.001). Results are mean percentage of WT total activity of the same day. (b) Effects of 4-day lithium treatment on spontaneous locomotor activity of GluR6 KO mice. Short-term lithium treatment did not significantly alter spontaneous locomotor activity of GluR6 KO mice. Data are percent of mean value of GluR6 KO mice treated with control food. Results are means±s.e.m. *Pp0.05.

WT 12 24 WT+Li * * GluR6 KO 20 GluR6 KO+Li 9 16 * * 6 12 * *

Frequency 8 3 Attack frequency 4

0 0 WT WT+Li Sniffing GluR6 KO Tall rattle Attack bite Side by side GluR6 KO+Li

150

125

100

75

50 GluR6 KO mice) Attack frequency 25 (% of control chow-treated 0 Control Lithium Figure 9 Chronic lithium dramatically decreased aggressive behavior in GluR6 KO mice. (a) Social interaction test: 4 weeks of lithium treatment caused a complete loss of tail rattling and attack bite events displayed by GluR6 KO mice toward their opponents from the same group (one-way ANOVA: tail rattling: F3,8 = 6.9, P < 0.05; attack bite F3,8 = 3.9, P = 0.05; sniffing:

F3,8 = 1.3, P = 0.3 (NS); side by side: F3,8 = 0.63, P = 0.6 (NS)). (b) Resident–intruder test: 4 weeks of lithium treatment caused a robust reduction in attacks displayed by GluR6 KO mice toward a naive male intruder of the same strain (one-way ANOVA:

F3,19 = 3.2, P < 0.05). Results are mean of percentage relative to WT±s.e.m. Li, lithium (chronic treatment). (c) Effects of 6 days of lithium treatment on measures of the resident–intruder test in GluR6 KO mice. Short-term lithium treatment did not significantly reduce attack frequencies of GluR6 KO mice (t14 = 1.171, P = 0.2611). Results are means±s.e.m. *P < 0.05. detected in the open-field test (Figure 8a), social inter- open-arm proportional measures of the elevated plus- action test (Figure 9a), resident–intruder test (Figure 9b) maze test in WT mice (duration: t10 = 0.9000, P = 0.3893; and center activity in the open-field test (Figure 10a). frequency: t10 = 0.435, P = 0.6728; distanced traveled: Chronic lithium treatment did not significantly alter t10 = 1.390, P = 0.1947).

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 866 1000 5000 900 4500 800 4000 700 3500 600 3000 500 2500 400 2000 300 WT mice) 1500 200 Center duration 1000 entries(% of control Frequency of center

chow-treated WT mice) 100 500 0 (% of control chow-treated 0 T +Li T +Li W T+Li 6 KO O W T+Li 6 KO O W 6 K W 6 K GluR GluR GluR GluR

250 200

200 150 150 100 100

Center duration 50 50 GluR6 KO mice) entries(% of control Frequency of center

0 (% of control chow-treated 0 chow-treated GluR6 KO mice) Control Lithium Control Lithium Figure 10 Chronic lithium treatment attenuated increased activity in anxiety-provoking regions. (a) Frequency of entries to the center of the arena during the first day of the open-field test. Four weeks of lithium treatment reduced the frequency of

entries to the center of the open-field arena in GluR6 KO mice (one-way ANOVA: F3,19 = 9.8, P < 0.001; post hoc Fisher LSD test: GluR6 KO vs GluR6 KO þ Li, WT þ Li or WT mice, P < 0.05; GluR6KO þ Li vs WT þ Li or WT mice, P = 0.01). (b) Duration of time spent in the center of the arena during the first day of open-field test. Four weeks of lithium treatment reduced the

amount of time spent in the center of the open-field arena in GluR6 KO mice (one-way ANOVA: F3,19 = 9.2, P < 0.001; post hoc Fisher LSD test: GluR6 KO vs GluR6 KO þ Li, WT þ Li or WT mice, P < 0.05; GluR6KO þ Li vs WT mice, P = 0.01). Results are means of percentage relative to WT±s.e.m. Li, lithium (chronic treatment). The effects of 5 days of lithium treatment on the increased activity in anxiety-provoking regions of the open-field test in GLuR6 KO mice were also studied. (c) The frequency of center entries of GluR6 KO mice was not reduced by short-term lithium treatment. (d) The time that GluR6 KO mice spent in the center of the arena was also not reduced by short-term lithium treatment. Results are means±s.e.m. *Pp0.05.

As noted previously, GluR6 KO mice were more P = 0.01) and time spent in the center of the open field

active on the locomotion measures (Figure 8a). (one-way ANOVA: F3,19 = 9.2, P < 0.001; post hoc Chronic (Figure 8a), but not short-term (Figure 8b), Fisher LSD test: GluR6 KO vs GluR6 KO þ Li, WT þ Li lithium treatment significantly lowered locomotor or WT mice, P < 0.05; GluR6KO þ Li vs WT mice, activity in GluR6 KO mice (two-way ANOVA: geno- P = 0.01).

type F3,57 = 647.7, P < 0.0001; day F2,57 = 1512, P = NS; Consistent with the findings described above, the genotype  day F6,57 = 0.8, P = NS; one-way ANOVA: GluR6 KO mice showed more proportional open-arm day 1: F3,19 = 7.7, P < 0.01; day 2: F3,19 = 25.7, P < 0.001; duration (t9 = 6.257, P = 0.0001), frequency (t9 = 4.681, day 3: F3,19 = 14.2, P < 0.001; post hoc Fisher LSD test: P = 0.0009) and traveled distance (t9 = 4.458, P = 0.0016). GluR6 KO vs WT and WT þ Li mice, P < 0.001; GluR6 Chronic or short-term lithium treatment did not KO vs GluR6 KO þ Li mice, P < 0.05). significantly alter measures of this test (Figure 11). As described above, GluR6 KO mice were aggres- In the forced swim test, the GluR6 KO mice sive (Figures 9a and b). Chronic (Figures 9a and b), exhibited less immobility compared to the WT mice but not short-term (Figure 9c), lithium treatment (Figure 12). Chronic lithium treatment reduced the significantly lowered tail rattle frequency (ANOVA, immobility of WT mice. Interestingly, lithium’s effects

F3,8 = 6.9, P < 0.05) and attack bite frequency in the on the GluR6 KO mice did not represent a generalized social interaction test (ANOVA, F3,8 = 3.9, P < 0.05), attenuation of activity, because lithium treatment and attack frequency in the resident–intruder test further reduced immobility in the forced swim test

(one-way ANOVA: F3,19 = 3.2, P < 0.05) in GluR6 KO (Figure 12) (two-way ANOVA: genotype F1,19 = 6.6, mice. P < 0.02; treatment F1,19 = 13.5, P < 0.01 with no inter- In this cohort of animals, GluR6 KO mice also action). displayed more activity in the center of the open-field arena (Figures 10a and b). Chronic (Figures 10a and Effects of ablation of GluR6 and chronic lithium b), but not short-term (Figures 10c and d), lithium treatment on cell membrane levels of ionotropic treatment significantly attenuated center entries (one- glutamatergic receptors

way ANOVA: F3,19 = 9.8, P < 0.001; post hoc Fisher To understand the potential involvement of other LSD test: GluR6 KO vs GluR6 KO þ Li, WT þ Li or WT ionotropic glutamatergic receptors in the behavioral mice, P < 0.05; GluR6 KO þ Li vs WT þ Li or WT mice, alterations of GluR6 KO mice, and the mechanisms

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 867 140 140 140 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 Changes in ratios of Changes in ratios of Changes in ratios of open vs total durations

0 0 open vs total distances 0

Control Lithium open vs total frequencies Control Lithium Control Lithium

160 160 160 140 140 140 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 Changes in ratios of Changes in ratios of Changes in ratios of open vs total distances open vs total durations 0 0 0 Control Lithium open vs total frequencies Control Lithium Control Lithium Figure 11 Effects of chronic and short-term lithium treatment on measures of the elevated plus maze test. Chronic (4 weeks, (a–c)) and short-term (4 days, (d–f)) lithium treatment did not significantly alter duration of time spent in the open arms of the maze (a and d), frequency of open-arm entries (b and e) and distance traveled in the open arms (c and f) in GluR6 KO mice. Results are means±s.e.m.

150 levels of GluR5 were not significantly altered by * GluR6 ablation and lithium treatment in the hippo- 125 campus and pre-FCX (Figures 13c and d). 100 The membrane levels of KA-2 receptors were 75 significantly reduced in GluR6 KO mice, but the reductions were not significantly reversed by chronic 50 lithium treatment in the hippocampus (Figures 14a

Immobility time (s) 25 and b) (one-way ANOVA: F2,22 = 168.34, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6 0 KO þ Li vs WT mice, P < 0.001; post hoc Fisher LSD T +Li W T+Li 6 KO W luR 6 KO test: GluR6 KO vs GluR6 KO þ Li mice, P = 0.98 (NS)) G luR G and in the FCX (one-way ANOVA: F2,22 = 84.14, Figure 12 Chronic lithium treatment further reduced P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6 KO and WT mice immobility time in the forced GluR6 KO þ Li vs WT mice, P < 0.001; post hoc Fisher swim test. Both GluR6 KO and WT mice reduced their LSD test: GluR6 KO vs GluR6 KO þ Li mice, P = 0.89 immobility time in the forced swim test by more than 60% (NS)). The cytosol levels of KA-2 receptors were not after chronic lithium treatment (two-way ANOVA: genotype significantly altered by GluR6 ablation and lithium F1,19 = 6.6, P < 0.02; treatment F1,19 = 13.5, P < 0.01 with no treatment in the hippocampus and pre-FCX (Figures interaction). Results are means of percentage relative to 14c and d). ± WT s.e.m. Li, lithium (chronic treatment). *P < 0.01. KARs subunit KA-1, the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2A, and the a-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) recep- underlying the behavioral effects of lithium in GluR6 tor subunits GluR1, GluR2 and GluR3 were not signi- KO mice, the cell membrane levels of ionotropic ficantly changed in the membrane fractions of the glutamatergic receptors were monitored in WT and hippocampus or FCX of the GluR6 KO mice, nor were GluR6 KO mice with or without chronic lithium the membrane fraction levels of these receptors affected treatment. The membrane levels of GluR5 were by lithium treatment (Table 1). significantly reduced in GluR6 KO mice, and the reductions were not significantly reversed by chronic Discussion lithium treatment in the hippocampus (one-way

ANOVA: F2,22 = 51.1, P < 0.001; post hoc Fisher LSD In this study, we were able to demonstrate, for the first test: GluR6 KO and GluR6 KO þ Li vs WT mice, time, that GluR6, a KAR subunit, plays a critical role P < 0.001; post hoc Fisher LSD test: GluR6 KO vs in the general control of diverse behavioral features, GluR6 KO þ Li mice, P = 0.80 (NS)) and in the FCX including hyperactivity, drive, aggressiveness, risk

(one-way ANOVA: F2,22 = 18.159, P < 0.001; post hoc taking (or less anxious behavior) and sensitivity to Fisher LSD test: GluR6 KO and GluR6KO þ Li vs WT psychostimulants. These features were demonstrated mice, P < 0.001; post hoc Fisher LSD test: GluR6 KO repeatedly in multiple tests (Figures 2–12). GluR6 vs GluR6 KO þ Li mice, P = 0.92 (NS)). The cytosol KO mice displayed these features without any

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 868 ug protein 6KO WT WT 6KO ug protein 6KO WT WT 6KO 510+Li 5 10 +Li

1.25 2 1.00 ** 0.75 1 0.50

0.25 FCX membrane GluR5 0.00 0

Hippocampal membrane GluR5 WT GluR6 KO GluR6 KOLi WT GluR6 KO GluR6 KOLi

ug proteinWT 6KO 6KO ug protein 6KO 6KO WT +Li 10 20 10 20 +Li 3 3

2 2

1 1 FCX cytosol GluR5 Hippocampal cytosol GluR5 0 0 WT GluR6 KO GluR6 KOLi WT GluR6 KO GluR6 KOLi Figure 13 The GluR5 subunit membrane expression levels were reduced in the hippocampus and FCX of GluR6 KO mice . (a) The GluR5 protein levels were reduced in the hippocampal membrane fraction of the GluR6 KO mice (one-way ANOVA:

F2,22 = 51.1, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6 KO þ Li vs WT mice, P < 0.001). Four weeks of chronic lithium treatment did not affect GluR5 protein level reduction in the hippocampal membrane fraction of the GluR6 KO mice (post hoc Fisher LSD test: GluR6 KO vs GluR6 KO þ Li mice, P = 0.80 (NS)). (b) The GluR5 protein levels were reduced in the

FCX membrane fraction of the GluR6 KO mice (one-way ANOVA: F2,22 = 18.159, P < 0.001; post hoc Fisher LSD test: GluR6 KO and GluR6KO þ Li vs WT mice, P < 0.001). Four weeks of chronic lithium treatment did not affect GluR5 protein level reduction in the FCX membrane fraction of the GluR6 KO mice (post hoc Fisher LSD test: GluR6 KO vs GluR6 KO þ Li mice, P = 0.92 (NS)). (c) The GluR5 protein levels were not reduced in the hippocampal cytosol fraction of GluR6 KO mice. Four weeks of chronic lithium treatment did not affect GluR5 levels in the hippocampal cytosolic fraction of GluR6 KO mice

(one-way ANOVA: F2,21 = 0.61, P = 0.55). (d) The GluR5 protein levels were not reduced in the FCX cytosol fraction of GluR6 KO mice. Four weeks of chronic lithium treatment did not affect the GluR5 levels in the FCX cytosolic fraction of GluR6

KO mice (one-way ANOVA: F2,18 = 2.63, P = 0.10 (NS)). FCX, frontal cortex; Li, lithium (chronic treatment). Results are mean±s.e.m. in arbitrary units (AU). *P < 0.001.

impairment of consciousness, sensory-motor func- synapses16 and, notably, are involved in long-term tion, learning or memory (Figure 1 and Mulle et potentiation induction at the hippocampus and the al.33,34). Furthermore, GluR6 ablation was associated amygdala.17 Presynaptic KARs control the release of with reduced membrane GluR5 or KA-2 receptors GABA in the hippocampus and the amygdala.16,18 (Figures 13, 14). Notably, this behavioral phenotype Evidence also suggests that KARs produce metabo- was highly selective for the GluR6 receptor, because tropic actions, such as interaction with G proteins, GluR5 ablation (another ionotropic KA receptor with induction of second messengers and the activation of many similarities to GluR6) was completely without PKA, PKC and MAP kinase. Through these actions, effect in any of these behavioral paradigms (Figures KARs are involved in the modulation of hippocampal 2–8). Membrane levels of other ionotropic glutama- inhibitory19 and excitatory postsynaptic currents,20 as tergic receptors tested remained unchanged in the well as pyramidal cell excitability.21,22 hippocampus and FCX of GluR6 KO mice (Table 1). As mentioned in the introduction, the GluR6 gene Thus, GluR6 appears to be uniquely able to control a is located on chromosome 6q21, a region reported subset of behavioral manifestations. by several groups to be linked with BPD.6–9 Moreover, KARs are known to function in the neuronal it has recently been shown that cortical expression circuitry of mood regulation. For instance, GluR5 is levels of GluR6 are lower in the cortex of BPD highly expressed in the amygdala, and GluR6 and KA- patients.10 Mania traditionally manifests as severe 1-2 are prominently expressed in the dentate gyrus mood elevation (either euphoria or irritability) and is and the CA3 region of the hippocampus.14,15 KARs accompanied by behavioral excitement, including regulate glutamate release at the mossy fiber to CA3 hyperactivity and psychomotor agitation, an increase

Molecular Psychiatry ncot i ihu;NMDA, lithium; Li, knockout; AMPA, MAGu10.76 GluR1 AMPA bevd hstedwsntafce yltimtetet(n-a NV:F ANOVA: (one-way treatment lithium by affected ( not mice was KO trend this GluR6 observed; of fraction membrane P FCX the in reduction P nrae A2poenlvl nteFXctslfato fGu6K iewsosre;ti rn a o fetdby affected not was trend this observed; was mice KO GluR6 of fraction cytosol F FCX ANOVA: the (one-way treatment in lithium levels protein KA-2 increased r mean are MAN10.17 NR1 NMDA lR KO GluR6 ramn i o fetK- rti ee euto ntehpoaplmmrn rcino lR Omc ( mice KO F GluR6 ANOVA: of (one-way fraction mice KO membrane KO GluR6 hippocampal GluR6 the vs of in KO fraction reduction GluR6 membrane level test: protein LSD KA-2 Fisher affect not did treatment type Receptor eut r eno rti eesi AU in levels protein of mean are Results treatment). (chronic Li ant A10.28 KA-1 Kainate 1 Table F ANOVA: (one-way mice KO GluR6 of fraction membrane hippocampal the in reduced were levels protein KA-2 14 Figure lR Omc,adaentafce yltimtreatment lithium by affected not are and mice, KO GluR6 .9(S) ( (NS)). 0.89 = 0.001; < a aio3hdoy5mty--sxzl;AOA nlsso aine U rirr nt;FX rna otx KO, cortex; frontal FCX, units; arbitrary AU, variance; of analysis ANOVA, -amino-3-hydroxy-5-methyl-4-isoxazole; ± oorpcguaaercpo uuismmrn xrsinlvl r o lee ntehpoapso C of FCX or hippocampus the in altered not are levels expression membrane subunits receptor glutamate Ionotropic othoc post uui TGu6K GluR6 KO GluR6 WT Subunit þ ...i rirr nt A) * (AU). units arbitrary in s.e.m. lR 0.53 GluR3 lR 0.76 GluR2 RA1.61 NR2A A2sbntmmrn xrsinlvl eerdcdi h ipcmu n C fGu6K ie ( mice. KO GluR6 of FCX and hippocampus the in reduced were levels expression membrane subunit KA-2 iv Tmice, WT vs Li c rn oadicesdK- rti eesi h ipcma yoo rcino lR Omc was mice KO GluR6 of fraction cytosol hippocampal the in levels protein KA-2 increased toward trend A )

ihrLDts:Gu6K n lR KO GluR6 and KO GluR6 test: LSD Fisher Hippocampal cytosol GluR5 Hippocampal membrane GluR5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 ± ± ± ± ± ± .70.51 0.07 .40.62 0.14 .11.06 0.21 .21.12 0.22 .40.19 0.04 .80.31 0.18 TGu6K GluR6KOLi GluR6KO WT 020 10 TGu6K GluR6KOLi GluR6KO WT gpoen6KO ug protein gpoen6KO ug protein P 510 N .0) orweso hoi ihu ramn i o fetteK- rti level protein KA-2 the affect not did treatment lithium chronic of weeks Four 0.001). < -methyl- ± ± ± ± ± ± ipcmu FCX Hippocampus .50.57 0.05 .10.59 0.11 .60.80 0.16 .61.33 0.16 .30.18 0.03 .90.36 0.09 2,18 +Li K WT 6KO ± D 1.37, = þ aprae T idtype. wild WT, -aspartate; P s.e.m. imice, Li TWT WT 0.001. < +Li KO P ± ± ± ± ± ± þ .8(S) C,fotlcre;L,ltim(hoi ramn) Results treatment). (chronic lithium Li, cortex; frontal FCX, (NS)). 0.28 = ** .50.27; 0.05 .90.49; 0.09 .41.06; 0.14 .51.55; 0.15 .30.07; 0.03 .80.18; 0.08 Li P 6KO .8(S) ( (NS)). 0.98 = NV F ANOVA 2,22 othoc post þ P P P P P P 84.14, = Shaltiel G behaviors mania-related regulates GluR6 .60.77 0.76 = .10.88 0.61 = .60.98 0.36 = .31.25 0.23 = .30.36 0.93 = .32.09 0.83 = iv Tmice, WT vs Li FCX cytosol KA-2 FCX membrane KA-2 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.0 0.5 1.0 1.5 2,22 b h A2poenlvl eerdcdi h FCX the in reduced were levels protein KA-2 The ) tal et ihrLDts:Gu6K sGu6KO GluR6 vs KO GluR6 test: LSD Fisher P 0.001; < TGu6K GluR6KOLi GluR6KO WT TGu6K GluR6KOLi GluR6KO WT TGu6K GluR6 KO GluR6 WT 5 gpoen6KO ug protein ± ± ± ± ± ± 2,21 .40.93 0.24 .90.92 0.19 .20.77 0.12 .81.02 0.88 .00.34 0.10 .61.68 0.46 020 10 gpoen6KO ug protein P 10 3.38, = othoc post .0) orweso hoi lithium chronic of weeks Four 0.001). < +Li P WT ± ± ± ± ± ± .5 N).( (NS)). 0.053 = TWT WT ihrLDts:Gu6K and KO GluR6 test: LSD Fisher .80.91 0.18 .50.87 0.15 .80.75 0.08 .62.09 0.66 .80.52 0.08 .42.36 0.34 6KO KO +Li 6KO ± ± ± ± ± ± þ .60.27; 0.16 .30.44; 0.13 .81.41; 0.08 .90.79; 0.59 .71.74; 0.07 .11.10; 0.31 Li d rn toward trend A ) NV F ANOVA 2,22 þ oeua Psychiatry Molecular 168.34, = othoc post imice, Li P P P P P P a 0.86 = 0.96 = 0.26 = 0.46 = 0.19 = 0.35 = The ) 2,22 869 GluR6 regulates mania-related behaviors G Shaltiel et al 870 in goal-directed activity and recklessness.39,40 Psycho- play a critical role in the control mechanism of some stimulants typically worsen manic symptoms,39,40 and facet of manic symptoms. lithium is the classic agent for alleviating those The cell surface expression levels of the different symptoms.39,40 Consistent with the genetic and post- KARs subunits have been shown to be regulated by mortem brain findings on GluR6, GluR6 KO mice alternative splicing, the process that determines the displayed behavioral alterations (Figures 2–12) that subunit trafficking properties from the ER (endoplas- appear to be phenocopies of the behavioral symptoms mic reticulum) to the membrane.43 The present of mania, although the mood and thought states of biochemical studies revealed a significant reduction these mice are obviously unknown. of both GluR5 and KA-2 subunits cell surface levels in Hyperactivity, increased risk-taking behavior and the hippocampus and the FCX of the GluR6 KO mice. aggressiveness per se can be observed in several Low levels of KA-2 subunit at the cell membrane in neuropsychiatric illnesses, such as ADHD, personal- the absence of the GluR6 subunit have been shown ity disorders, organic brain disorders and brain in vitro by several other groups (reviewed in Jaskolski trauma. Therefore, the pathophysiological mechan- et al.43). KA-2 subunits and the alternative splice isms underlying each of these three symptoms are variant GluR5c are known to fail to reach the plasma very diverse and might not be directly linked to those membrane because of the presence of a retention for manic symptoms. This is consistent with motif in their sequences that prevents them from both animal studies and online data (http://www. exiting the ER unless they are assembled in hetero- informatics.jax.org) reporting that a single-related meric KARs.44–46 GluR5-a and -b splice variants are behavior can result from the mutation of a variety of targeted to the plasma membrane but at low levels genes. However, comorbidity of the three symptoms is in homomeric forms. GluR6a splice variant contains more common in manic patients, and all three symp- a specific forwarding trafficking domain, making it a toms respond to lithium treatment in manic patients. key subunit that, upon assembling in heteromeric There are no sufficient or convincing clinical data to receptors, promotes the cell surface expression of all support the notion that lithium therapy is effective in other ER-retained KARs subunits.44 Thus, consistent the treatment of ADHD or aggression. GluR6 KO mice with findings from in vitro studies, we found a co-displayed hyperactivity, risk-taking behavior and tremendous decrease in KARs from the cell surface aggression (Figures 2–12). Chronic lithium treatment, in the hippocampus and the FCX of the GluR6 KO at least, partially alleviated these behavioral altera- mice. These decreases are likely due to GluR6 tions in GluR6 KO mice (Figure 8–10). Therefore, ablation-associated trafficking impairments of GluR5 from a phenomenal similarity point of view, the and KA-2 receptors. current data collectively support the notion that The question of whether GluR6 ablation-associated GluR6 contributes to control mechanisms related to changes of GluR5 and KA-2 contribute to the beha- facets of manic symptoms. vioral displays of GluR6 KO mice remains unan- We have chosen to use the term phenocopy, as this swered. One of the limitations of the present study is emphasizes apparent phenomenological similarity, that the role of KA-2 in behavioral regulation was not which can result from the same or different under- directly investigated. On the other hand, GluR5 KO lying mechanism(s). In our opinion, the term ‘model’ mice did not display similar behavioral alterations to is far more stringent, requiring additional criteria, those observed in GluR6 KO mice (Figures 2–8). including phenomenological similarity, treatment Chronic lithium treatment produced significant beha- response similarity, induction mechanistic similarity vioral effects in GluR6 KO mice; however, it did not and pathophysiological similarity.24,41,42 Despite in- reverse reductions of GluR5 and KA-2 in the hippo- tensive research and intriguing findings, such as campal and frontal cortical membrane fractions. The changes in GluR6 levels reported in the post-mortem effects of chronic lithium treatment on membrane cerebral cortex of bipolar patients, the induction levels of GluR5 and KA-2 in other brain regions need mechanism and the symptom pathophysiology of to be investigated. mania are still largely unknown. Although it is It is interesting to note that recent clinical studies plausible that GluR6 dysfunction is involved in the show that ketamine, a non-competitive NMDA recep- pathophysiology of manic symptoms, further clinical tor antagonist, produces rapid-onset antidepressant investigations are needed to establish this link. effects in depressed patients.47,48 An animal study Bipolar disorder has a unique cycling feature. further showed that ketamine produces antidepres- Although this study was initiated, in part, by the sant-like effects in animal models and that the effect of genetic findings that linked GluR6 polymorphism to ketamine can be blocked by NBQX (1,2,3,4-tetrahydro- the increased risk of BPD, the data presented here do 6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide), not directly address whether GluR6 functional altera- an AMPA and KA receptor antagonist.30 Chronic tions change the risk for BPD. Furthermore, the beha- lithium treatment decreased the levels of GluR1 in vioral patterns of GluR6 KO mice as well as GluR6 membrane fraction, whereas treatment with imipra- ablation-associated behavioral displays cannot be mine, lamotrigine and riluzole had the opposite viewed as a model of BPD because the model would effect. These data suggest that NMDA and AMPA lack the spontaneous cycling feature. Rather, the receptors are crucially involved in regulating mood- current data support the notion that GluR6 might related behaviors. Interestingly, the hippocampal and

Molecular Psychiatry GluR6 regulates mania-related behaviors G Shaltiel et al 871 prefrontal-cortical membrane levels of GluR1-3, NR1 2 Baum A, Akula N, Cabenero M, Cardona I, Corona W, Klemens B and NR2A were not altered by GluR6 ablation and et al. A genome-wide association study implicates diacylglycerol were not affected by chronic lithium treatment in kinase eta (DGKH) and several other genes in the etiology of bipolar disorder. Mol Psychiatry 2007; 13: 197–207. GluR6 KO mice. The plausible explanation is that 3 Kato T. Molecular genetics of bipolar disorder and depression. the synaptic machinery responsible for behavioral Psychiatry Clin Neurosci 2007; 61: 3–19. excitement abnormalities differs from that used by 4 The Wellcome Trust Case Control Consortium. Genome-wide antidepressant agents to produce desired behavioral association study of 14 000 cases of seven common diseases and outcomes; furthermore, ionotropic glutamatergic 3000 shared controls. Nature 2007; 447: 661–678. 5 Buervenich S, Detera-Wadleigh SD, Akula N, Thomas CJM, receptors appear to play a role in regulating behavior Kassem L, Rezvani A et al. Fine mapping on chromosome 6q in in a brain regional-specific manner. the NIMH Genetics Initiative bipolar pedigrees (abstract 1882). These findings place GluR6 into a small group of Annual Meeting of The American Society of Human Genetics. molecules that are known to significantly contribute Los Angeles, CA, 2003 (http://www.genetics.faseb.org/genetics/ to the behavioral control mechanisms related to ashg03s/index.shtml). 10,49 6 Dick DM, Foroud T, Flury L, Bowman ES, Miller MJ, Rau NL et al. different facets of manic symptoms. These mole- Genomewide linkage analyses of bipolar disorder: a new sample of cules include GSK-3b (glycogen synthase kinase 3b),50 250 pedigrees from the National Institute of Mental Health CLOCK,51 ERK1 (also known as MAPK3)26 and mutant Genetics Initiative. Am J Hum Genet 2003; 73: 107–114. mtDNA polymerase.52 Mice with genetic alterations of 7 McQueen MB, Devlin B, Faraone SV, Nimgaonkar VL, Sklar P, Smoller JW et al. Combined analysis from eleven linkage studies of these molecules display some common phenotypes, bipolar disorder provides strong evidence of susceptibility loci on 26,50,51,53,54 including hyperactivity in multiple tests, 6q and 8q. Am J Hum Genet 2005; 77: 582–595. sensitivity to psychostimulants and rewards,26,51,53,55,56 8 Schulze TG, Buervenich S, Badner JA, Steele CJ, Detera-Wadleigh less anxiety or more risk taking in anxiety-related SD, Dick DM et al. Loci on chromosomes 6q and 6p interact to tests,26,51 less behavioral despair or more drive in increase susceptibility to bipolar affective disorder in the national 26,50,51 51,52 institute of mental health genetics initiative pedigrees. Biol immobility tests and circadian alterations. Psychiatry 2004; 56: 18–23. 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